Phenotype-Independent Isolation of Interspecies Saccharomyces Hybrids by Dual-Dye Fluorescent Staining and Fluorescence-Activated Cell Sorting

Abstract

Interspecies hybrids of Saccharomyces species are found in a variety of industrial environments and often outperform their parental strains in industrial fermentation processes. Interspecies hybridization is therefore increasingly considered as an approach for improvement and diversification of yeast strains for industrial application. However, current hybridization methods are limited by their reliance on pre-existing or introduced selectable phenotypes. This study presents a high-throughput phenotype-independent method for isolation of interspecies Saccharomyces hybrids based on dual dye-staining and subsequent mating of two strains, followed by enrichment of double-stained hybrid cells from a mating population by fluorescence-activated cell sorting (FACS). Pilot experiments on intra-species mating of heterothallic haploid S. cerevisiae strains showed that 80% of sorted double-stained cells were hybrids. The protocol was further optimized by mating an S. cerevisiae haploid with homothallic S. eubayanus spores with complementary selectable phenotypes. In crosses without selectable phenotype, using S. cerevisiae and S. eubayanus haploids derived from laboratory as well as industrial strains, 10 to 15% of double-stained cells isolated by FACS were hybrids. When applied to rare mating, sorting of double-stained cells consistently resulted in about 600-fold enrichment of hybrid cells. Mating of dual-stained cells and FACS-based selection allows efficient enrichment of interspecies Saccharomyces hybrids within a matter of days and without requiring selectable hybrid phenotypes, both for homothallic and heterothallic strains. This strategy should accelerate the isolation of laboratory-made hybrids, facilitate research into hybrid heterosis and offer new opportunities for non-GM industrial strain improvement and diversification.

Keywords: FACS; Saccharomyces eubayanus × Saccharomyces cerevisiae hybrids; heterosis; lager beer brewing; marker-free mating; non-GMO.

FIGURE 1

Intra-species mating of S. cerevisiae strains CEN.PK113-5A (MATa URA3 his3-Δ1 leu2-3,112 trp1-289) and IMK439 (MATα HIS3 TRP1 LEU2 ura3Δ::KanMX). (A) Fluorescence contour plots of unstained CEN.PK113-5A, CEN.PK113-5A stained with CellTrace^TM CFSE, IMK439 stained with CellTrace^TM Violet, and of the mating culture after 18, 24, and 42 h. The indicated gated areas were used for sorting cells, event rates of each gate are indicated as a percentage of total cell counts. (B) Microscope image (400×) of zygotes sorted from the double-stained population (C+V+) after 42 h of mating. (C) Percentage of cells able to grow on synthetic medium without auxotrophy-complementing supplements in different populations sorted by FACS, as indicated in (A).

FIGURE 2

Optimization of interspecies hybridization between haploid S. eubayanus and S. cerevisiae strains. (A) Overview of the optimized protocol for interspecies spore-to-cell mating. (B) Fluorescence contour plots of mating cultures of stained CBS 12357 spores and IMK439 cells after 30 h of mating on YPD and YPT. The gated areas were used for sorting cells, event rates of each gate are indicated as a percentage. (C) Percentages of cells in the double-stained population able to grow on SM+G418 after 3.5, 7, 24, and 30 h of incubation on YPT (black) and YPD (white). Mating on YPT was only assessed at 7 and 30 h.

FIGURE 3

Enrichment of interspecies hybrids without selectable phenotypes from CBS 12357 (S. eubayanus, sporulated) and CEN.PK113-7D (MATa) and from CDFM21L.1 (S. eubayanus, sporulated) and Ale28 (S. cerevisiae, sporulated). (A) Fluorescence contour plots of mating cultures between CBS 12357 (CFSE) × CEN.PK113-7D (Violet) and CDFM21L.1 (CFSE) × Ale28 (Violet). Gated areas were used for sorting cells, event rates of each gate are indicated as a percentage of the total population size. (B) Flow cytometric quantification of the genome content of constructed hybrids using SYTOX Green staining. S. cerevisiae strains CEN.PK113-7D and CEN.PK122 were used as a haploid and diploid control, respectively. (C) Multiplex PCR amplification of the S. cerevisiae specific MEX67 gene (150 bp) and the S. eubayanus specific FSY1 gene (228 bp) in single-cell isolates of the double-stained populations from CBS 12357 × CEN.PK113-7D and CDFM21L.1 × Ale28 mating cultures. For CBS 12357 × CEN.PK113-7D, 4 of the 22 tested isolates are shown. Genomic DNA of S. cerevisiae Ale26, S. eubayanus CDFM21L.1 and S. cerevisiae × S. eubayanus IMS0408 were used as controls. Hybrid isolates are indicated by arrows. L: Generuler 50 bp DNA Ladder.